A fundamental step in replication of enveloped viruses is the generation of viral envelope. While most enveloped viruses obtain their envelope by budding from cellular compartments, nucleocytoplasmic large DNA viruses (NCLDVs), including poxvirus, asfarvirus and mimivirus, acquire their primary envelope through assembly of open-ended, crescent membranes derived from endoplasmic reticulum (ER). This highly unusual process of membrane biogenesis has been enigmatic for over half a century. Recent studies with vaccinia virus (VACV) have identified five viral proteins to be individually essential for this process. These so-called viral membrane assembly proteins (VMAPs) are thought to be involved in generating and/or stabilizing scission of ER membranes, but their mechanisms of action are unknown. We have made sustained contributions to the understanding of VACV membrane biogenesis process for over a decade, including the identification of VACV A6 as a VMAP and the determination of the structures of A6 and another VACV VMAP, H7. We found that H7 binds phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P) and that A6 C- terminal domain (A6-C) traps multiple lipids with a membrane bilayer-like configuration, revealing a novel molecular modality for enclosing the lipid bilayer. Moreover, we uncovered an essential interaction between A6 and H7 by employing a novel experimental viral evolution approach. These findings led to our innovative hypothesis that H7 and A6 bind respectively to the hydrophilic head and the hydrophobic acyl tail of phospholipids, working in concert to generate and/or stabilize open-ended membrane sheets. With a long-term goal of fully elucidating the poxvirus membrane biogenesis process, our current objective is to test our novel hypothesis and determine the molecular mechanism by which A6 and H7 coordinate in membrane scission and remodeling. Aim 1. To determine the mechanism by which A6 coordinates with H7 in binding lipids. Aim 2. To determine the roles of A6 and H7 in generating membrane scissions. The proposed study on poxvirus VMAPs will not only elucidate a key viral replication step for antiviral development but also provide insights into the process of cellular membrane scission and remodeling.